Abbreviations
TFA, trifluoroacetic acid PA0 Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid PA0 Dibal, diisobutyl aluminum hydride PA0 NBS, N-bromosuccinimide PA0 HPLC, high-performance liquid chromatography PA0 Caged P.sub.i, 1-(o-nitrophenyl)-ethyl phosphate PA0 BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid PA0 EDTA, ethylenediaminetetraacetic acid PA0 EGTA, ethylenebis(oxyethylenenitrilo)tetraacetic acid PA0 Tes, (N-tris[hyroxymethyl]methyl-2-aminoethanesulfonic acid PA0 HDTA, N-hyroxyethylethylenediaminetriacefic acid
Calcium is an important second messenger for a wide variety of physiological and biochemical processes such as muscle contraction, neurotransmitter release, ion channel gating, exocytosis, etc. The essential role of Ca.sup.2+ release and sequestration in intracellular communication has also been recently highlighted by the growing appreciation of the importance of inositol phospholipid metabolism in signaling. A technique for the controlled, localized, and rapid increase in [Ca.sup.2+ ] would provide a tool which would enable the study of the kinetic, regulatory, and structural mechanisms of such processes to be undertaken. Two approaches to this problem have been taken (reviewed in ref. 1 ). The first, developed by Tsien and co-workers, involves reducing the Ca.sup.2+ -buffering capacity of a BAPTA derivative by decreasing the electron donating capacity of one of the coordinating ligands on illumination following the photoexpulsion of a small molecule from the chelator. This strategy has led to two readily available photosensitive buffers, nitr-5 and nitr-7 (2).
Our approach is conceptually different in that we have designed photosensitive derivatives of chelators with known high affinity for Ca.sup.2+, which upon illumination are bifurcated, producing two moieties with known low affinity, thus the bound Ca.sup.2+ is released (cf. Scheme 1).
DM-nitrophen is a (now) commercially available photosensitive derivative of EDTA (3,4) representative of this approach which has found wide application during the last several years as a caged Ca (5-14) and caged Mg (15). The distinct advantage of nitr-5 and nitr-7 compared to DM-nitrophen is that they are Ca.sup.2+ -selective chelators whereas DM-nitrophen has chelation properties similar to EDTA from which it is derived. The comparative advantages of DM-nitrophen are that its Ca.sup.2+ affinity is very high before photolysis and very low after photolysis, thus ensuring a good photochemical yield of liberated Ca.sup.2+.
In our U.S. Pat. No. 4,981,985, the entire disclosure of which is incorporated by reference herein, we disclosed the synthesis of photolabile chelators for multivalent cations. The disclosure included the method of synthesizing photolabile chelators as EDTA and EGTA derivatives to be used in caging multivalent cations. The chelators chelate the cations forming non-biologically active compounds. Upon irradiation, the chelated compound cleaves with the subsequent cleaved remainders having a substantially lower affinity for the chelated cation. Large mounts of cation are thus rapidly released and the effect of such concentration jumps on the biological system can be accurately studied.
We now describe a new photosensitive Ca.sup.2+ chelator, called nitrophenyl-EGTA or NP-EGTA (1), that binds Ca.sup.2+ selectively with high affinity (80 nM), which upon photolysis is bifurcated producing iminodiacetic acid photoproducts (see Scheme 1 ) with a 12,500-fold lower affinity for Ca.sup.2+. This new compound possesses the desired properties of Ca.sup.2+ selectivity in combination with a rapid high photochemical yield of liberated Ca.sup.2+.
Accordingly, a need exists for a caged calcium which selectively binds Ca.sup.2+ with high affinity and releases it rapidly upon photolysis.